Fig. 7. Glucosidase II is not packaged into COPII vesicles in vitro and strains lacking a cycling Erv41p-Erv46p complex have a glycoprotein processing defect. (A) Microsomes from a strain expressing Rot2p-3HA (SOY01117) were incubated without (–) or with (+) purified COPII subunits in the presence of GTP and an ATP regeneration system. Membranes from 10% of the total reaction (T) and vesicle fractions were collected by centrifugation, resolved on a polyacrylamide gel and immunoblotted. (B) ³⁵S-labelled factor pheromone precursor was translocated into semi-intact cells. Membranes were solubilized and glycoproteins precipitated on concanavalin A Sepharose beads. Bound radiolabelled protein was eluted from the beads, resolved on polyacrylamide gels and detected by phosphorimaging. Fully core glycosylated factor pheromone precursor is indicated (gpf). The lower bands represent partially glycosylated species. Arrowheads mark the band representing incompletely trimmed gpf. Semi-intact cells were prepared from wild-type (wt, FY834), rot2 (CBY1087), cwh41 (CBY1086), an erv41 strain containing empty vector pRS314 (CBY1036), or an erv41 strain containing the ERV41 gene in vector pRS314 (CBY1037). (C) Same procedure as in B, but semi-intact cells were prepared from an erv41 strain containing a plasmid which encodes a truncated version of ERV41 lacking C-terminal residues 340-352 (CBY1038), or from erv41 strains transformed with pRS314 plasmids containing alanine scan ERV41 mutants (CBY1074-1080, CBY1089-1096, CBY1158 and CBY1159, respectively).